Multiphoton microscopy assessment of the structure and variability changes of dermal connective tissue in vulvar lichen sclerosus: A pilot study.

In this article, we offer a novel classification of progressive changes in the connective tissue of dermis in vulvar lichen sclerosus (VLS) relying on quantitative assessment of the second harmonic generation (SHG) signal received from formalin fixed and deparaffinized tissue sections. We formulate criteria for distinguishing four degrees of VLS development: Initial-Mild-Moderate-Severe. Five quantitative characteristics (length and thickness type I Collagen fibers, Mean SHG signal intensity, Skewness and Coherence SHG signal) are used to describe the sequential degradation of connective tissue (changes in the structure, orientation, shape and density of collagen fibers) up to the formation of specific homogeneous masses. Each of the degrees has a characteristic set of quantitatively expressed features. We focus on the identification and description of early, initial changes of the dermis as the least specific. The results obtained by us and the proposed classification of the degrees of the disease can be used to objectify the dynamics of tissue changes during treatment.

Multiphoton tomography in differentiation of morphological and molecular subtypes of breast cancer: A quantitative analysis.

In this study multiphoton tomography, based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) was used to visualize both the extracellular matrix and tumor cells in different morphological and molecular subtypes of human breast cancer. It was shown, that quantified assessment of the SHG based imaging data has great potential to reveal differences of collagen quantity, organization and uniformity in both low- and highly- aggressive invasive breast cancers. The values of quantity and uniformity of the collagen fibers distribution were significantly higher in low-aggressive breast cancer compared to the highly-aggressive subtypes, while the value representing collagen organization was lower in the former type. Additionally, it was shown, that TPEF detection of elastin fibers and amyloid protein may be used as a biomarker of detection the low-aggressive breast cancer subtype. Thus, TPEF/SHG imaging offers the possibility of becoming a useful tool for the rapid diagnosis of various subtypes of breast cancer during biopsy as well as for the intraoperative determinination of tumor-positive resection margins.

In vivo assessment of functional and morphological alterations in tumors under treatment using OCT-angiography combined with OCT-elastography.

Emerging methods of anti-tumor therapies require new approaches to tumor response evaluation, especially enabling label-free diagnostics and in vivo utilization. Here, to assess the tumor early reaction and predict its long-term response, for the first time we apply in combination the recently developed OCT extensions - optical coherence angiography (OCA) and compressional optical coherence elastography (OCE), thus enabling complementary functional/microstructural tumor characterization. We study two vascular-targeted therapies of different types, (1) anti-angiogenic chemotherapy (ChT) and (2) photodynamic therapy (PDT), aimed to indirectly kill tumor cells through blood supply injury. Despite different mechanisms of anti-angiogenic action for ChT and PDT, in both cases OCA demonstrated high sensitivity to blood perfusion cessation. The new method of OCE-based morphological segmentation revealed very similar histological structure alterations. The OCE results showed high correlation with conventional histology in evaluating percentages of necrotic and viable tumor zones. Such possibilities make OCE an attractive tool enabling previously inaccessible in vivo monitoring of individual tumor response to therapies without taking multiple biopsies.

Can "Indirect" Contact Laser Surgery be Used for Fluorescence-Image Guided Tumor Resections? Preliminary Results.

Ensuring the complete removal of tumor tissue is the main challenge during resection operations. Recently, a technique of "indirect" contact laser surgery has been developed. In this study we assess the possibility of using such surgery for fluorescence image-guided tumor resection. Mouse colon adenocarcinoma CT-26 cells stably expressing the fluorescent protein mKate-2 was used as the tumor model. Resections of the tumor nodes were performed with either a scalpel blade, a laser scalpel with a bare tip, or a laser scalpel with a strongly absorbing coating on the fiber tip. Tumor-positive resection margins were detected using an IVIS Spectrum fluorescence imaging system. After tumor resection with the scalpel blade over half of the animals needed one additional resection to remove residual tumor cells. Animals in this group showed tumor recurrence within 7 days. Fluorescence imaging of the tumor bed, performed after resection to assess the presence of tumor cell clusters, was sufficiently effective only with a bloodless resection. The laser scalpels both with the bare tip and with the strongly absorbing coating on the tip provided such bloodless tumor resection in contact mode. Fewer animals required additional resections when the bare tipped scalpel was used and this also resulted in a reduction in tumor recurrence. After resections were carried out with the laser scalpel with the strongly absorbing coating on the tip, fluorescence was detected in the operative field and this led to undertaking additional resections, although subsequent investigation suggested that this was "false" fluorescence, resulting from the effects of the scalpel rather than the presence of residual tumor cells. The method of laser resection with a strongly absorbing coating on the tip therefore did not appear to demonstrate definite advantages over laser resection with a bare tip when removing tumors.

OCT-guided laser hyperthermia with passively tumor-targeted gold nanoparticles.

The goal of this study is the development of a method of local laser hyperthermia with gold nanoparticles under noninvasive optical monitoring of nanoparticle accumulation in tumor tissue in vivo. Bifunctional plasmon resonant nanoparticles that are optimal for OCT diagnostics and laser heating at the wavelength of 810 nm were used in the study. The OCT examination showed that the accumulation of gold nanoparticles in the tumor invading into skin was maximal 4-5 h after intravenous injection. It was demonstrated that nanoparticle accumulation in tumor allowed more local heating and enhanced thermal sensitivity of tumor tissue. Laser hyperthermia that heated tumor up to 44-45 °C at maximum nanoparticle accumulation induced apoptotic death of tumor cells and inhibited tumor growth by 104% on the 5th day after treatment.